The orphan nuclear receptor steroidogenic factor 1 (SF-1) regulates the differentiation and function of endocrine glands. Although SF-1 is constitutively active in cell-based assays, it is not known whether this transcriptional activity is modulated by ligands. Here, we describe the 1.5 angstroms crystal structure of the SF-1 ligand binding domain in complex with an LXXLL motif from a coregulator protein. The structure reveals the presence of a phospholipid ligand in a surprisingly large pocket (approximately 1600 angstroms3), with the receptor adopting the canonical active conformation. The bound phospholipid is readily exchanged and modulates SF-1 interactions with coactivators. Mutations designed to reduce the size of the SF-1 pocket or to disrupt hydrogen bonds with the phospholipid abolish SF-1/coactivator interactions and significantly reduce SF-1 transcriptional activity. These findings provide evidence that SF-1 is regulated by endogenous ligands and suggest an unexpected relationship between phospholipids and endocrine development and function.

Mineralocorticoid receptor (MR) controls sodium homeostasis and blood pressure through hormone binding and coactivator recruitment. Here, we report a 1.95 A crystal structure of the MR ligand binding domain containing a single C808S mutation bound to corticosterone and the fourth LXXLL motif of steroid receptor coactivator-1 (SRC1-4). Through a combination of biochemical and structural analyses, we demonstrate that SRC1-4 is the most potent MR binding motif and mutations that disrupt the MR/SRC1-4 interactions abolish the ability of the full-length SRC1 to coactivate MR. The structure also reveals a compact steroid binding pocket with a unique topology that is primarily defined by key residues of helices 6 and 7. Mutations swapping a single residue at position 848 from helix H7 between MR and glucocorticoid receptor (GR) switch their hormone specificity. Together, these findings provide critical insights into the molecular basis of hormone binding and coactivator recognition by MR and related steroid receptors.

Constitutive androstane receptor (CAR) induces xenobiotic, bilirubin, and thyroid hormone metabolism as a heterodimer with the retinoid X receptor (RXR). Unlike ligand-dependent nuclear receptors, CAR is constitutively active. Here, we report the heterodimeric structure of the CAR and RXR ligand binding domains (LBDs), which reveals an unusually large dimerization interface and a small CAR ligand binding pocket. Constitutive CAR activity appears to be mediated by the compact nature of the CAR LBD that displays several unique features including a shortened AF2 helix and helix H10, which are linked by a two-turn helix that normally adopts an extended loop in other receptors, and an extended helix H2 that stabilizes the canonical LBD fold by packing tightly against helix H3. These structural observations provide a molecular framework for understanding the atypical transcriptional activation properties of CAR.

Constitutive androstane receptor (CAR) induces xenobiotic, bilirubin, and thyroid hormone metabolism as a heterodimer with the retinoid X receptor (RXR). Unlike ligand-dependent nuclear receptors, CAR is constitutively active. Here, we report the heterodimeric structure of the CAR and RXR ligand binding domains (LBDs), which reveals an unusually large dimerization interface and a small CAR ligand binding pocket. Constitutive CAR activity appears to be mediated by the compact nature of the CAR LBD that displays several unique features including a shortened AF2 helix and helix H10, which are linked by a two-turn helix that normally adopts an extended loop in other receptors, and an extended helix H2 that stabilizes the canonical LBD fold by packing tightly against helix H3. These structural observations provide a molecular framework for understanding the atypical transcriptional activation properties of CAR.

The functional interaction between the orphan nuclear receptors small heterodimer partner (SHP) and liver receptor homolog 1 (LRH-1), where SHP binds to LRH-1 and represses its constitutive transcriptional activity, is crucial for regulating genes involved in cholesterol homeostasis. Here, we report structural and biochemical analyses of the LRH-1͞SHP interaction. The crystal structure and modeling studies of the LRH-1 ligand-binding domain bound to either of the two LXXLL-related motifs of SHP show that the receptor undergoes conformational changes to accommodate the SHP docking and reveal key residues that determine the potency and selectivity of SHP binding. Through a combination of mutagenesis and binding studies, we demonstrate that only the second SHP LXXLL motif is required for repressing LRH-1, and this motif displays a strong preference for binding to LRH-1 over the closely related receptor steroidogeneic factor 1 (SF-1). Structural comparisons indicate that this binding selectivity is determined by residues flanking the core LXXLL motifs. These results establish a structural model for understanding how SHP interacts with LRH-1 to regulate cholesterol homeostasis and provide new insights into how nuclear receptor͞coregulator selectivity is achieved.steroidogeneic factor 1 ͉ bile acids ͉ coactivators ͉ corepressors L iver receptor homolog 1 (LRH-1, NR5A2) is an orphan nuclear receptor that activates an array of genes responsible for development of endodermal organs such as liver, intestine, and pancreas (reviewed in ref. 1). LRH-1 also plays a central role in lipid homeostasis by regulating genes involved in bile acid synthesis, reverse cholesterol transport, and metabolism of lipoprotein complexes (2-6). In the nuclear receptor superfamily, LRH-1 is most homologous to steroidogeneic factor 1 (SF-1, NR5A1), which is essential for sex differentiation and development of adrenals and gonads (7,8). LRH-1 and SF-1 share a highly conserved DNA-binding domain (DBD, Ͼ90% identity) and a moderately conserved ligand-binding domain (LBD, 56% identity). In contrast to most other nuclear receptors that function as dimers, LRH-1 and SF-1 bind with high affinity as monomers to a conserved consensus DNA site found in the promoters of target genes (9). The high degree of similarity in the DBDs of LRH-1 and SF-1 suggests that their different biological activities are contributed in part by distinct structural features in their LBDs, which recruit specific cofactors to regulate transcription.LRH-1 seems to be a constitutively active transcription factor in that it activates many reporters in the absence of any exogenous ligands. The crystal structure of the mouse LRH-1 LBD reveals a sandwich fold of four layers of helices instead of the three layers observed for many other receptors (10). Despite the absence of any ligand in the large ligand-binding pocket, the C-terminal activation helix (AF-2) of LRH-1 is packed in an active conformation. The constitutive LRH-1 activity is proposed to be the result of the stabilization o...